巴西橡胶树6个PP2C家族基因成员分子信息学与抗旱功能分析

PP2C是一类丝氨酸/苏氨酸残基蛋白磷酸酶,在高等植物ABA信号途径中起着重要的作用。为阐明巴西橡胶树中PP2C基因的结构与功能,本研究通过生物信息学方法,从橡胶树转录组数据库中鉴定并获得6个PP2C家族基因,均含有PP2CD、F1和F2亚族。通过qRT-PCR技术对6个PP2C家族基因进行了干旱处理下的差异表达分析,发现6个基因都不同程度上响应橡胶树干旱胁迫。本研究为探究PP2C基因在橡胶树抗干旱反应机制提供了理论依据。     

Type 2C protein phosphatases in fungi

Type 2C Ser/Thr phosphatases are a remarkable class of protein phosphatases, which are conserved in eukaryotes and involved in a large variety of functional processes. Unlike in other Ser/Thr phosphatases, the catalytic polypeptide is not usually associated with regulatory subunits, and functional specificity is achieved by encoding multiple isoforms. For fungi, most information comes from the study of type 2C protein phosphatase (PP2C) enzymes in Saccharomyces cerevisiae, where seven PP2C-encoding genes (PTC1 to -7) with diverse functions can be found. More recently, data on several Candida albicans PP2C proteins became available, suggesting that some of them can be involved in virulence. In this work we review the available literature on fungal PP2Cs and explore sequence databases to provide a comprehensive overview of these enzymes in fungi.     

Plant protein phosphatases 2C: from genomic diversity to functional multiplicity and importance in stress management

Protein phosphatases (PPs) counteract kinases in reversible phosphorylation events during numerous signal transduction pathways in eukaryotes. Type 2C PPs (PP2Cs) represent the major group of PPs in plants, and recent discovery of novel abscisic acid (ABA) receptors (ABARs) has placed the PP2Cs at the center stage of the major signaling pathway regulating plant responses to stresses and plant development. Several studies have provided deep insight into vital roles of the PP2Cs in various plant processes. Global analyses of the PP2C gene family in model plants have contributed to our understanding of their genomic diversity and conservation, across plant species. In this review, we discuss the genomic and structural accounts of PP2Cs in plants. Recent advancements in their interaction paradigm with ABARs and sucrose nonfermenting related kinases 2 (SnRK2s) in ABA signaling are also highlighted. In addition, expression analyses and important roles of PP2Cs in the regulation of biotic and abiotic stress responses, potassium (K⁺) deficiency signaling, plant immunity and development are elaborated. Knowledge of functional roles of specific PP2Cs could be exploited for the genetic manipulation of crop plants. Genetic engineering using PP2C genes could provide great impetus in the agricultural biotechnology sector in terms of imparting desired traits, including a higher degree of stress tolerance and productivity without a yield penalty.     

Protein phosphatase 2C (PP2C) function in higher plants

In the past few years, molecular cloning studies have revealed the primary structure of plant protein serine/threonine phosphatases. Two structurally distinct families, the PP1/PP2A family and the PP2C family, are present in plants as well as in animals. This review will focus on the plant PP2C family of protein phosphatases. Biochemical and molecular genetic studies in Arabidopsis have identified PP2C enzymes as key players in plant signal transduction processes. For instance, the ABI1/ABI2 PP2Cs are central components in abscisic acid (ABA) signal transduction. Arabidopsis mutants containing a single amino acid exchange in ABI1 or ABI2 show a reduced response to ABA. Another member of the PP2C family, kinase-associated protein phosphatase (KAPP), appears to be an important element in some receptor-like kinase (RLK) signalling pathways. Finally, an alfalfa PP2C acts as a negative regulator of a plant mitogen-activated protein kinase (MAPK) pathway. Thus, the plant PP2Cs function as regulators of various signal transduction pathways.     

The Arabidopsis phosphatase PP2C49 negatively regulates salt tolerance through inhibition of AtHKT1;1

Type 2C protein phosphatases (PP2Cs) are the largest protein phosphatase family. PP2Cs dephosphorylate substrates for signaling in Arabidopsis, but the functions of most PP2Cs remain unknown. Here, we characterized PP2C49 (AT3G62260, a Group G PP2C), which regulates Na⁺ distribution under salt stress and is localized to the cytoplasm and nucleus. PP2C49 was highly expressed in root vascular tissues and its disruption enhanced plant tolerance to salt stress. Compared with wild type, the pp2c49 mutant contained more Na⁺ in roots but less Na⁺ in shoots and xylem sap, suggesting that PP2C49 regulates shoot Na⁺ extrusion. Reciprocal grafting revealed a root‐based mechanism underlying the salt tolerance of pp2c49. Systemic Na⁺ distribution largely depends on AtHKT1;1 and loss of function of AtHKT1;1 in the pp2c49 background overrode the salt tolerance of pp2c49, resulting in salt sensitivity. Furthermore, compared with plants overexpressing PP2C49 in the wild‐type background, plants overexpressing PP2C49 in the athtk1;1 mutant background were sensitive to salt, like the athtk1;1 mutants. Moreover, protein–protein interaction and two‐voltage clamping assays demonstrated that PP2C49 physically interacts with AtHKT1;1 and inhibits the Na⁺ permeability of AtHKT1;1. This study reveals that PP2C49 negatively regulates AtHKT1;1 activity and thus determines systemic Na⁺ allocation during salt stress.     

The protein phosphatase 2C (PP2C) superfamily: detection of bacterial homologues.

A thorough sequence analysis of the various members of the eukaryotic protein serine/threonine phosphatase 2C (PP2C) family revealed the conservation of 11 motifs. These motifs could be identified in numerous other sequences, including fungal adenylate cyclases that are predicted to contain a functionally active PP2C domain, and a family of prokaryotic serine/threonine phosphatases including SpoIIE. Phylogenetic analysis of all the proteins indicates a widespread sequence family for which a considerable number of isoenzymes can be inferred.     

Molecular character of a phosphatase 2C (PP2C) gene relation to stress tolerance in Arabidopsis thaliana

Protein phosphatases type 2C (PP2Cs) from group A, which includes the ABI1/HAB1 and PP2CA branches, are key negative regulators of ABA signaling. HAI-1 gene had been shown to affect both seed and vegetative responses to ABA, which is one of PP2Cs clade A in Arabidopsis thaliana. Transgenic plants containing pHAI-1::GUS (β-glucuronidase) displayed GUS activity existing in the vascular system of leave veins, stems and petioles. Green fluorescent protein fused HAI-1 (HAI-1-GFP) was found in the nucleus through transient transformation assays with onion epidermal cells. The water-loss assays indicated the loss-of-function mutants did not show symptoms of wilting and they had still turgid green rosette leaves. The assays of seed germination by exogenous ABA and NaCl manifested that the loss-of-function mutants displayed higher insensitivity than wild-type plants. Taken together, the final results suggest that the HAI-1 (AT5G59220) encoded a nuclear protein and it can be highly induced by ABA and wound in Arabidposis, the stress-tolerance phenotype showed a slightly improvement when HAI-1 gene was disrupted.     

Genome-wide analysis and expression profiling of PP2C clade D under saline and alkali stresses in wild soybean and <Emphasis Type="Italic">Arabidopsis</Emphasis>

Protein phosphatase 2Cs (PP2Cs) belong to the largest protein phosphatase family in plants. Some members have been described as being negative modulators of plant growth and development, as well as responses to hormones and environmental stimuli. However, little is known about the members of PP2C clade D, which may be involved in the regulation of signaling pathways, especially in response to saline and alkali stresses. Here, we identified 13 PP2C orthologs from the wild soybean (Glycine soja) genome. We examined the sequence characteristics, chromosome locations and duplications, gene structures, and promoter cis-elements of the PP2C clade D genes in Arabidopsis and wild soybean. Our results showed that GsPP2C clade D (GsAPD) genes exhibit more gene duplications than AtPP2C clade D genes. Plant hormone and abiotic stress-responsive elements were identified in the promoter regions of most PP2C genes. Moreover, we investigated their expression patterns in roots, stems, and leaves. Quantitative real-time PCR analyses revealed that the expression levels of representative GsPP2C and AtPP2C clade D genes were significantly influenced by alkali and salt stresses, suggesting that these genes might be associated with or directly involved in the relevant stress signaling pathways. Our results established a foundation for further functional characterization of PP2C clade D genes in the future.     

Expression profiles and genomic organisation of group A protein phosphatase 2C genes in Brassica oleracea

We investigated the expression profiles and genomic organisation of the ABA‐responsive genes encoding protein phosphatases 2C (PP2C, group A members) in Brassica oleracea to better understand their functional and genetic relations. Gene expression profiling of drought responsive genes in B. oleracea and Arabidopsis thaliana revealed significant differences in the gene expression pattern of a key regulator of ABA signalling—ABI1 PP2C. This finding prompted us to study genetic relations within the PP2Cs group A in the Brassica species. Twenty homologous B. oleracea sequences were identified and characterised as putative PP2C group A members. Phylogenetic analysis revealed that the B. oleracea homologues were closely related to the particular members of the A. thaliana PP2C. The genetic analysis corroborated the presence of two to three gene copies in B. oleracea in comparison to the nine unique PP2C genes in the A. thaliana genome. Gene expression analyses showed significant differences in PP2C gene expression pattern in B. oleracea. Our results indicate that PP2C‐based drought stress signalling in B. oleracea has evolved distinctly. Different reactions of particular B. oleracea PP2C genes to drought stress and ABA treatment indicate low conservation of gene expression patterns and functional divergence between B. oleracea and A. thaliana homologous genes.     

A type 2C protein phosphatase FgPtc3 is involved in cell wall integrity, lipid metabolism, and virulence in Fusarium graminearum

Type 2C protein phosphatases (PP2Cs) play important roles in regulating many biological processes in eukaryotes. Currently, little is known about functions of PP2Cs in filamentous fungi. The causal agent of wheat head blight, Fusarium graminearum, contains seven putative PP2C genes, FgPTC1, -3, -5, -5R, -6, -7 and -7R. In order to investigate roles of these PP2Cs, we constructed deletion mutants for all seven PP2C genes in this study. The FgPTC3 deletion mutant (ΔFgPtc3-8) exhibited reduced aerial hyphae formation and deoxynivalenol (DON) production, but increased production of conidia. The mutant showed increased resistance to osmotic stress and cell wall-damaging agents on potato dextrose agar plates. Pathogencity assays showed that ΔFgPtc3-8 is unable to infect flowering wheat head. All of the defects were restored when ΔFgPtc3-8 was complemented with the wild-type FgPTC3 gene. Additionally, the FgPTC3 partially rescued growth defect of a yeast PTC1 deletion mutant under various stress conditions. Ultrastructural and histochemical analyses showed that conidia of ΔFgPtc3-8 contained an unusually high number of large lipid droplets. Furthermore, the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Quantitative real-time PCR assays showed that basal expression of FgOS2, FgSLT2 and FgMKK1 in the mutant was significantly higher than that in the wild-type strain. Serial analysis of gene expression in ΔFgPtc3-8 revealed that FgPTC3 is associated with various metabolic pathways. In contrast to the FgPTC3 mutant, the deletion mutants of FgPTC1, FgPTC5, FgPTC5R, FgPTC6, FgPTC7 or FgPTC7R did not show aberrant phenotypic features when grown on PDA medium or inoculated on wheat head. These results indicate FgPtc3 is the key PP2C that plays a critical role in a variety of cellular and biological functions, including cell wall integrity, lipid and secondary metabolisms, and virulence in F. graminearum.     

Evolution of the Metazoan Protein Phosphatase 2C Superfamily

Members of the protein phosphatase 2C (PP2C) superfamily are Mg²⁺/Mn²⁺-dependent serine/threonine phosphatases, which are essential for regulation of cell cycle and stress signaling pathways in cells. In this study, a comprehensive genomic analysis of all available metazoan PP2C sequences was conducted. The phylogeny of PP2C was reconstructed, revealing the existence of 15 vertebrate families which arose following a series of gene duplication events. Relative dating of these duplications showed that they occurred in two active periods: before the divergence of bilaterians and before vertebrate diversification. PP2C families which duplicated during the first period take part in different signaling pathways, whereas PP2C families which diverged in the second period display tissue expression differences yet participate in similar signaling pathways. These differences were found to involve variation of expression in tissues which show higher complexity in vertebrates, such as skeletal muscle and the nervous system. Further analysis was performed with the aim of identifying the functional domains of PP2C. The conservation pattern across the entire PP2C superfamily revealed an extensive domain of more than 50 amino acids which is highly conserved throughout all PP2C members. Several insertion or deletion events were found which may have led to the specialization of each PP2C family. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Hector Musto]     

Catalytic mechanism and kinase interactions of ABA-signaling PP2C phosphatases

Abscisic acid (ABA) is an essential hormone that controls plant growth, development and responses to abiotic stresses. ABA signaling is mediated by type 2C protein phosphatases (PP2Cs), including HAB1 and ABI2, which inhibit stress-activated SnRK2 kinases and whose activity is regulated by ABA and ABA receptors. Based on biochemical data and our previously determined crystal structures of ABI2 and the SnRK2.6–HAB1 complex, we present the catalytic mechanism of PP2C and provide new insight into PP2C–SnRK2 interactions and possible roles of other SnRK2 kinases in ABA signaling.     

Substrate-dependent metal preference of PPM1H, a cancer-associated protein phosphatase 2C: comparison with other family members

Protein phosphatase 2C (PP2C) family is characterized by requirement of metal cation for phosphatase activity. We previously established that PPM1H is a cancer-associated member of the PP2C family. Here we further characterized the phosphatase activity of PPM1H, focusing on its dependence on metal cation. PPM1H possesses the potential to dephosphorylate p-nitrophenyl phosphate (pNPP), casein and phosphopeptides. Interestingly, PPM1H shows the metal preference that is varied depending on the substrate (substrate-dependent metal preference); PPM1H prefers Mn²⁺ when pNPP or phosphopeptides is used as a substrate. Meanwhile, a preference for Mg²⁺ is displayed by PPM1H with casein as a substrate. When both cations are added to the reaction, the degree of the effect is always closer to that with Mn²⁺ alone, irrespective of the substrate. This preponderance of Mn²⁺ is explained by its greater affinity for PPM1H than Mg²⁺. From the literature the substrate-dependent metal preference appears to be shared by other PP2Cs. According to the crystal structure, a binuclear metal center of PP2C plays an important role for coordinating the substrate and nucleophilic waters in the active site. Therefore, the differences in the size, preferred geometry and coordination requirements between two metals, in relation to the substrate, may be responsible for this intriguing property.     

植物PP2C蛋白磷酸酶ABA信号转导及逆境胁迫调控机制研究进展

蛋白磷酸酶(protein phosphatase,PP)是蛋白质可逆磷酸化调节机制中的关键酶,而PP2C磷酸酶是一类丝氨酸/苏氨酸残基蛋白磷酸酶,是高等植物中最大的蛋白磷酸酶家族,包含76个家族成员,广泛存在于生物体中。迄今为止,在植物体内已经发现了4种PP2C蛋白磷酸酶。蛋白激酶和蛋白磷酸酶协同催化蛋白质可逆磷酸化,在植物体内信号转导和生理代谢中起着重要的调节作用,蛋白质的磷酸化几乎存在于所有的信号转导途径中。大量研究表明,PP2Cs参与多条信号转导途径,包括PP2C参与ABA调控,对干旱、低温、高盐等逆境胁迫的响应,参与植物创伤和种子休眠或萌发等信号途径,其调控机制不同,但酶催化活性都依赖于Mg2+或Mn2+的浓度。植物PP2C蛋白的C端催化结构域高度保守,而N端功能各异。文中还综述了高等植物PP2C的分类、结构、ABA受体与PP2Cs蛋白互作、PP2C基因参与ABA信号途径以及其他逆境信号转导途径的研究进展。     

Gene Descent, Duplication, and Horizontal Transfer in the Evolution of Glutamyl- and Glutaminyl-tRNA Synthetases

In translation, separate aminoacyl-tRNA synthetases attach the 20 different amino acids to their cognate tRNAs, with the exception of glutamine. Eukaryotes and some bacteria employ a specific glutaminyl-tRNA synthetase (GlnRS) which other Bacteria, the Archaea (archaebacteria), and organelles apparently lack. Instead, tRNA^Gln is initially acylated with glutamate by glutamyl-tRNA synthetase (GluRS), then the glutamate moiety is transamidated to glutamine. Lamour et al. [(1994) Proc Natl Acad Sci USA 91:8670–8674] suggested that an early duplication of the GluRS gene in eukaryotes gave rise to the gene for GlnRS—a copy of which was subsequently transferred to proteobacteria. However, questions remain about the occurrence of GlnRS genes among the Eucarya (eukaryotes) outside of the ``crown' taxa (animals, fungi, and plants), the distribution of GlnRS genes in the Bacteria, and their evolutionary relationships to genes from the Archaea. Here, we show that GlnRS occurs in the most deeply branching eukaryotes and that putative GluRS genes from the Archaea are more closely related to GlnRS and GluRS genes of the Eucarya than to those of Bacteria. There is still no evidence for the existence of GlnRS in the Archaea. We propose that the last common ancestor to contemporary cells, or cenancestor, used transamidation to synthesize Gln-tRNA^Gln and that both the Bacteria and the Archaea retained this pathway, while eukaryotes developed a specific GlnRS gene through the duplication of an existing GluRS gene. In the Bacteria, GlnRS genes have been identified in a total of 10 species from three highly diverse taxonomic groups: Thermus/Deinococcus, Proteobacteria γ/β subdivision, and Bacteroides/Cytophaga/Flexibacter. Although all bacterial GlnRS form a monophyletic group, the broad phyletic distribution of this tRNA synthetase suggests that multiple gene transfers from eukaryotes to bacteria occurred shortly after the Archaea–eukaryote divergence.     

A hypermorphic mutation in the protein phosphatase 2C HAB1 strongly affects ABA signaling in Arabidopsis

Protein phosphatases of the 2C family (PP2C) function in the regulation of several signaling pathways from prokaryotes to eukaryotes. In Arabidopsis thaliana, the HAB1 PP2C is a negative regulator of the stress hormone abscisic acid (ABA) signaling. Here, we show that plants expressing a mutant form of HAB1 in which Gly246 was mutated to Asp (G246D) display strong ABA insensitive phenotypes. Our results indicate that the G246D mutation has a hypermorphic rather than a dominant negative effect. The data suggest that this mutation localized in a conserved motif in the PP2C catalytic domain could be used in other PP2Cs to reveal their biological functions.     

Overexpression of PP2A‐C5 that encodes the catalytic subunit 5 of protein phosphatase 2A in Arabidopsis confers better root and shoot development under salt conditions

Protein phosphatase 2A (PP2A) is an enzyme consisting of three subunits: a scaffolding A subunit, a regulatory B subunit and a catalytic C subunit. PP2As were shown to play diverse roles in eukaryotes. In this study, the function of the Arabidopsis PP2A‐C5 gene that encodes the catalytic subunit 5 of PP2A was studied using both loss‐of‐function and gain‐of‐function analyses. Loss‐of‐function mutant pp2a‐c5‐1 displayed more impaired growth during root and shoot development, whereas overexpression of PP2A‐C5 conferred better root and shoot growth under different salt treatments, indicating that PP2A‐C5 plays an important role in plant growth under salt conditions. Double knockout mutants of pp2a‐c5‐1 and salt overly sensitive (sos) mutants sos1‐1, sos2‐2 or sos3‐1 showed additive sensitivity to NaCl, indicating that PP2A‐C5 functions in a pathway different from the SOS signalling pathway. Using yeast two‐hybrid analysis, four vacuolar membrane chloride channel (CLC) proteins, AtCLCa, AtCLCb, AtCLCc and AtCLCg, were found to interact with PP2A‐C5. Moreover, overexpression of AtCLCc leads to increased salt tolerance and Cl^? accumulation in transgenic Arabidopsis plants. These data indicate that PP2A‐C5‐mediated better growth under salt conditions might involve up‐regulation of CLC activities on vacuolar membranes and that PP2A‐C5 could be used for improving salt tolerance in crops.     

Evidence of independent gene duplications during the evolution of archaeal and eukaryotic family B DNA polymerases

Eukaryotes and archaea both possess multiple genes coding for family B DNA polymerases. In animals and fungi, three family B DNA polymerases, alpha, delta, and epsilon, are responsible for replication of nuclear DNA. We used a PCR-based approach to amplify and sequence phylogenetically conserved regions of these three DNA polymerases from Giardia intestinalis and Trichomonas vaginalis, representatives of early-diverging eukaryotic lineages. Phylogenetic analysis of eukaryotic and archaeal paralogs suggests that the gene duplications that gave rise to the three replicative paralogs occurred before the divergence of the earliest eukaryotic lineages, and that all eukaryotes are likely to possess these paralogs. One eukaryotic paralog, epsilon, consistently branches within archaeal sequences to the exclusion of other eukaryotic paralogs, suggesting that an epsilon-like family B DNA polymerase was ancestral to both archaea and eukaryotes. Because crenarchaeote and euryarchaeote paralogs do not form monophyletic groups in phylogenetic analysis, it is possible that archaeal family B paralogs themselves evolved by a series of gene duplications independent of the gene duplications that gave rise to eukaryotic paralogs.